Increasing Cellular Lipid Production by Increasing the Activity of Diacylglycerol Acyltransferase and Decreasing the Activity of Triacylglycerol Lipase

ABSTRACT

Disclosed are methods and compositions for increasing the triacylglycerol content of a cell by up-regulating diacylglycerol acyltransferase and down-regulating triacylglycerol lipase. In some embodiments, a DGA1 protein is expressed and a native TGL3 gene is knocked out, thereby increasing the synthesis of triacylglycerol and decreasing its consumption, respectively.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 15/307,518, filed Oct. 28, 2016, now U.S. Pat. No. 10,443,047, which claims the benefit of PCT/US2015/028760 filed May 1, 2015, which claims priority from U.S. Provisional Application No. 61/987,098, filed May 1, 2014, and U.S. Provisional Application No. 62/090,169, filed Dec. 10, 2014, which are incorporated herein by reference in their entireties for all purposes.

BACKGROUND

Lipids have multiple industrial applications, including in the cosmetic and food industries, as well as serving as precursors for biodiesel and biochemical production. Microbial lipids are produced by many oleaginous organisms, including the well-characterized yeast Yarrowia lipolytica. Lipid yield in oleaginous organisms can be increased by up-regulation or down-regulation or deletion of genes implicated in the lipid pathway. For example, it was reported that up-regulation of native Y. lipolytica DGA1 significantly increased lipid yield and productivity (Metabolic Engineering 15:1-9 (2013)).

Y. lipolytica DGA1 is a type 2 diacylglycerol acyltransferase encoded by the Y. lipolytica diacylglycerol acyltransferase gene DGAT2. DGA1 is one of the key enzymes in the lipid pathway, and it is involved in the final step of triacylglycerol (TAG) synthesis. Triacylglycerols are the major form of storage lipids in Y. lipolytica. Recent data suggests that DGA1 efficiency may be a significant factor for high levels of lipid accumulation in oleaginous organisms (Metabolic Engineering 15:1-9 (2013)). Additionally, DGA1 genes from other species can be introduced into the host genome and have a significant effect on lipid production and composition. For example, other oleaginous yeast, such as Rhodosporidium toruloides and Lipomyces starkeyi, are able to accumulate significantly more lipids compared to wild type Y. lipolytica strains. It was demonstrated that overexpression in Y. lipolytica of DGA1 from organisms with higher native lipid production levels had a greater effect on Y. lipolytica lipid production than overexpression of native Y. lipolytica DGA1 (U.S. Ser. No. 61/943,664; incorporated by reference). Despite efforts to increase lipid yield in Y. lipolytica by overexpression of DGA1 from Mortierella alpine, no significant effect on lipid production levels has been reported (U.S. Pat. No. 7,198,937; incorporated by reference). The deletion of genes involved in the breakdown of lipids or in pathways that draw flux away from lipid biosynthesis has also been studied. Dulermo et al. demonstrated that the deletion of the triacylglycerol lipase gene TGL3 nearly doubled the total lipid content accumulated by Y. lipolytica (Biochimica Biophysica Acta 1831:1486-95 (2013)). The TGL3 protein is one of two intracellular lipases responsible for the first step of triacylglycerol degradation in Y. lipolytica.

SUMMARY

In some embodiments, the invention provides a transformed oleaginous cell comprising a first genetic modification, wherein said first genetic modification increases the activity of a native diacylglycerol acyltransferase or encodes at least one copy of a diacylglycerol acyltransferase gene native to the oleaginous cell or from a different species, and a second genetic modification, wherein said second genetic modification decreases the activity of a triacylglycerol lipase in the oleaginous cell.

In some aspects, the invention provides a method of increasing the lipid content of a cell comprising transforming a parent cell with a first nucleic acid and a second nucleic acid, wherein said first nucleic acid increases the activity of a native diacylglycerol acyltransferase or comprises a diacylglycerol acyltransferase gene and said second nucleic acid decreases the activity of a triacylglycerol lipase. Alternatively, the same nucleic acid could embody both elements described above. Thus, the invention also provides a method of increasing the lipid content of a cell comprising transforming a parent cell with a nucleic acid, wherein said nucleic acid decreases the activity of a triacylglycerol lipase in the cell, and said nucleic acid comprises a diacylglycerol acyltransferase gene or increases the activity of a native diacylglycerol acyltransferase.

Further, in some aspects, the invention provides a method of increasing the triacylglycerol content of a cell comprising: (a) providing a cell, comprising (i) a first genetic modification, wherein said first genetic modification increases the activity of a native diacylglycerol acyltransferase, or encodes at least one copy of a diacylglycerol acyltransferase gene native to the oleaginous cell or from a different species; and (ii) a second genetic modification that decreases the activity of a triacylglycerol lipase in the cell; (b) growing said cell under conditions whereby the first genetic modification is expressed, thereby producing a triacylglycerol; and (c) optionally recovering the triacylglycerol.

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiments described herein are not intended as limitations on the scope of the invention. These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description, drawings, and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a map of the pNC243 construct used to overexpress the diacylglycerol acyltransferase DGA1 gene NG66 in Y. lipolytica strain NS18 (obtained from ARS Culture Collection, NRRL # YB 392). Vector pNC243 was linearized by PacI/NotI restriction digest before transformation. “2u ori” denotes the S. cerevisiae origin of replication from the 2 μm circle plasmid; “pMB1 ori” denotes the E. coli pMB1 origin of replication from the pBR322 plasmid; “AmpR” denotes the bla gene used as a marker for selection with ampicillin; “PR2” denotes the Y. lipolytica GPD1 promoter −931 to −1; “NG66” denotes the native Rhodosporidium toruloides DGA1 cDNA synthesized by GenScript; “TER1” denotes the Y. lipolytica CYC1 terminator 300 base pairs after stop; “PR22” denotes the S. cerevisiae TEF1 promoter −412 to −1; “NG3” denotes the Streptomyces noursei Nat1 gene used as a marker for selection with nourseothricin; “TER2” denotes the S. cerevisiae CYC1 terminator 275 base pairs after stop; and “Sc URA3” denotes the S. cerevisiae URA3 auxotrophic marker for selection in yeast.

FIG. 2 is a graph depicting results from a fluorescence-based lipid assay for Y. lipolytica strain NS421, which is a triacylglycerol lipase knockout strain, and Y. lipolytica strain NS377, which is a triacylglycerol lipase knockout strain that overexpresses the diacylglycerol acyltransferase DGA1 gene NG66.

FIG. 3 is a graph depicting results from a gas chromatography analysis of Y. lipolytica strain NS281, which overexpresses the diacylglycerol acyltransferase DGA1 gene NG66, and Y. lipolytica strain NS377, containing a triacylglycerol lipase knockout and overexpressing the diacylglycerol acyltransferase DGA1 gene NG66.

FIG. 4 depicts a map of the pNC327 construct used to overexpress the NG112 gene (C. purpurea DGA2) in Y. lipolytica. Vector pNC327 was linearized by a PacI/AscI restriction digest before transformation. “2u ori” denotes the S. cerevisiae origin of replication from the 2 μm circle plasmid; “pMB1 ori” denotes the E. coli pMB1 origin of replication from the pBR322 plasmid; “AmpR” denotes the bla gene used as a marker for selection with ampicillin; “PR3” denotes the Y. lipolytica TEF1 promoter −406 to +125; “NG112” denotes the C. purpurea DGA2 gene synthetized by GenScript; “TER1” denotes the Y. lipolytica CYC1 terminator 300 bp after stop; “PR1” denotes the Y. lipolytica TEF1 promoter −406 to −1; “NG76” denotes the Streptoalloteichus hindustanus BLE gene used as marker for selection with Zeocin; “TER7” denotes the Y. lipolytica TEF1 terminator 400 bp after stop; and “Sc URA3” denotes the S. cerevisiae URA3 auxotrophic marker for selection in yeast.

FIG. 5 comprises three panels, labeled pane (A), (B), and (C). The figure depicts lipid accumulation measured by a fluorescence-based assay or a percentage of the dry cell weight as determined by gas chromatography for Yarrowia lipolytica strains NS297, NS281, NS450, NS377, and NS432. NS297 expresses an additional copy of Y. lipolytica DGA1; NS281 expresses Rhodosporidium toruloides DGA1; NS450 expresses R. toruloides DGA1 and Claviceps purpurea DGA2; NS377 expresses R. toruloides and carries a deletion of Y. lipolytica TGL3; NS432 expresses R. toruloides DGA1 and C. purpurea DGA2 and carries a deletion of Y. lipolytica TGL3. In panel (A), strains were analyzed by fluorescence assay after 96 hours of fermentation in a 48-well plate where two or three transformants were analyzed for each construct. In panel (B), strains were analyzed by fluorescence assay and gas chromatography after 96 hours of fermentation in 50-mL flasks. In panel (C), strains were analyzed by gas chromatography after 140 hours of fermentation in 1-L bioreactors. Data for NS281, NS377, and NS432 are averages obtained from duplicate bioreactor fermentations. Data for NS450 represents the value obtained from a single bioreactor fermentation.

DETAILED DESCRIPTION Overview

Disclosed are methods and compositions for creating transformed oleaginous cells with increased triacylglycerol content. Disrupting the yeast triacylglycerol lipase gene TGL3 eliminates a pathway that depletes triacylglycerol content. Additionally, overexpressing the diacylglycerol acyltransferase DGA1 increases the amount of protein that can synthesize triacylglycerol. Therefore, combining DGA1 overexpression with a TGL3 deletion could be an attractive approach to further increase a cell's triacylglycerol content; however, the manipulation of proteins that affect a metabolic pathway is unpredictable at best. Cells that suppress the effects of a genetic modification frequently possess a selective advantage over those that acquire the desired trait. Thus, cells that display a desired phenotype are often difficult to engineer.

Disclosed is the successful combination of TGL3 deletion and DGA1 overexpression to increase a cell's triacylglycerol content. TGL3 deletion and the concomitant expression of the R. toruloides DGA1 diacylglycerol acyltransferase in Y. lipolytica strains resulted in a higher triacylglycerol content than strains carrying a single genetic modification.

Definitions

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

The term “activity” refers to the total capacity of a cell to perform a function. For example, a genetic modification that decreases the activity of a triacylglycerol lipase in a cell may reduce the amount of triacylglycerol lipase in a cell or reduce the efficiency of triacylglycerol lipase. A triacylglycerol lipase knockout reduces the amount of triacylglycerol lipase in the cell. Alternatively, a mutation to a triacylglycerol lipase gene may reduce the efficiency of its triacylglycerol lipase protein product with little effect on the amount of cellular triacylglycerol lipase. Mutations that reduce the efficiency of triacylglycerol lipase may affect the active site, for example, by changing one or more active site residues; they may impair the enzyme's kinetics, for example, by sterically blocking substrates or products; they may affect protein folding or dynamics, for example, by reducing the proportion of properly-folded enzymes; they may affect protein localization, for example, by preventing the lipase from localizing to lipid particles; or they may affect protein degradation, for example, by adding one or more protein cleavage sites or by adding one or more residues or amino acid sequences that target the protein for proteolysis. These mutations affect coding regions. Mutations that decrease triacylglycerol lipase activity may instead affect the transcription or translation of the gene. For example, mutation to a triacylglycerol lipase enhancer or promoter can reduce triacylglycerol lipase activity by reducing its expression. Mutating or deleting the non-coding portions of a triacylglycerol lipase gene, such as its introns, may also reduce transcription or translation. Additionally, mutations to the upstream regulators of a triacylglycerol lipase may affect triacylglycerol lipase activity; for example, the over-expression of one or more repressors may decrease triacylglycerol lipase activity, and a knockout or mutation of one or more activators may similarly decrease triacylglycerol lipase activity. A genetic modification that increases the activity of a diacylglycerol acyltransferase in a cell may increase the amount of triacylglycerol acyltransferase in a cell or increase the efficiency of diacylglycerol acyltransferase. For example, the genetic modification may simply insert an additional copy of diacylglycerol acyltransferase into the cell such that the additional copy is transcribed and translated into additional functional diacylglycerol acyltransferase. The added diacylglycerol acyltransferase gene can be native to the host organism or from a different organism. Alternatively, mutating or deleting the non-coding portions of a native diacylglycerol acyltransferase gene, such as its introns, may also increase translation. A native diacylglycerol acyltransferase gene can be altered by adding a new promoter that causes more transcription. Similarly, enhancers may be added to the diacylglycerol acyltransferase gene that increase transcription, or silencers may be mutated or deleted from the diacylglycerol acyltransferase gene to increase transcription. Mutations to a native gene's coding region might also increase diacylglycerol acyltransferase activity, for example, by producing a protein variant that does not interact with inhibitory proteins or molecules. The over-expression of one or more activators may increase diacylglycerol acyltransferase activity by increasing the expression of a diacylglycerol acyltransferase protein, and a knockout or mutation of one or more repressors may similarly increase diacylglycerol acyltransferase activity.

The term “biologically-active portion” refers to an amino acid sequence that is less than a full-length amino acid sequence, but exhibits at least one activity of the full length sequence. For example, a biologically-active portion of a diacylglycerol acyltransferase may refer to one or more domains of DGA1 or DGA2 having biological activity for converting acyl-CoA and diacylglycerol to triacylglycerol. Biologically-active portions of a DGA1 include peptides or polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the DGA1 protein, e.g., the amino acid sequence as set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61 which include fewer amino acids than the full length DGA1, and exhibit at least one activity of a DGA1 protein. Similarly, biologically-active portions of a DGA2 protein include peptides or polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the DGA2 protein, e.g., the amino acid sequence as set forth in SEQ ID NOs: 29, 31, 33, 35, 37, 39, 63, 65, 67, 69, 71, 73, or 75, which include fewer amino acids than the full length DGA2, and exhibit at least one activity of a DGA2 protein. Biologically-active portions of a DGA3 protein include peptides or polypeptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the DGA3 protein, e.g., the amino acid sequence as set forth in SEQ ID NOs: 83 or 85, which include fewer amino acids than the full length DGA3, and exhibit at least one activity of a DGA3 protein. A biologically-active portion of a diacylglycerol acyltransferase may comprise, for example, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, or 696 amino acids. Typically, biologically active portions comprise a domain or motif having the catalytic activity of converting acyl-CoA and diacylglycerol to triacylglycerol. A biologically active portion of a DGA1 protein can be a polypeptide which is, for example, 278 amino acids in length.

The term “DGAT1” refers to a gene that encodes a type 1 diacylglycerol acyltransferase protein, such as a gene that encodes a DGA2 protein.

The term “DGAT2” refers to a gene that encodes a type 2 diacylglycerol acyltransferase protein, such as a gene that encodes a DGA1 protein.

The term “DGAT3” refers to a gene that encodes a type 2 diacylglycerol acyltransferase protein, such as a gene that encodes a DGA3 protein.

“Diacylglyceride,” “diacylglycerol,” and “diglyceride,” are esters comprised of glycerol and two fatty acids.

The terms “diacylglycerol acyltransferase” and “DGA” refer to any protein that catalyzes the formation of triacylglycerides from diacylglycerol. Diacylglycerol acyltransferases include type 1 diacylglycerol acyltransferases (DGA2), type 2 diacylglycerol acyltransferases (DGA1), and all homologs that catalyze the above-mentioned reaction.

The terms “diacylglycerol acyltransferase, type 1” and “type 1 diacylglycerol acyltransferases” refer to DGA2 and DGA2 orthologs.

The terms “diacylglycerol acyltransferase, type 2” and “type 2 diacylglycerol acyltransferases” refer to DGA1 and DGA1 orthologs.

The terms “diacylglycerol acyltransferase, type 3” and “type 3 diacylglycerol acyltransferases” refer to DGA3 and DGA3 orthologs.

The term “domain” refers to a part of the amino acid sequence of a protein that is able to fold into a stable three-dimensional structure independent of the rest of the protein.

The term “drug” refers to any molecule that inhibits cell growth or proliferation, thereby providing a selective advantage to cells that contain a gene that confers resistance to the drug. Drugs include antibiotics, antimicrobials, toxins, and pesticides.

“Dry weight” and “dry cell weight” mean weight determined in the relative absence of water. For example, reference to oleaginous cells as comprising a specified percentage of a particular component by dry weight means that the percentage is calculated based on the weight of the cell after substantially all water has been removed.

The term “encode” refers to nucleic acids that comprise a coding region, portion of a coding region, or compliments thereof. Both DNA and RNA may encode a gene. Both DNA and RNA may encode a protein.

The term “exogenous” refers to anything that is introduced into a cell. An “exogenous nucleic acid” is a nucleic acid that entered a cell through the cell membrane. An exogenous nucleic acid may contain a nucleotide sequence that exists in the native genome of a cell and/or nucleotide sequences that did not previously exist in the cell's genome. Exogenous nucleic acids include exogenous genes. An “exogenous gene” is a nucleic acid that codes for the expression of an RNA and/or protein that has been introduced into a cell (e.g., by transformation/transfection), and is also referred to as a “transgene.” A cell comprising an exogenous gene may be referred to as a recombinant cell, into which additional exogenous gene(s) may be introduced. The exogenous gene may be from the same or different species relative to the cell being transformed. Thus, an exogenous gene can include a native gene that occupies a different location in the genome of the cell or is under different control, relative to the endogenous copy of the gene. An exogenous gene may be present in more than one copy in the cell. An exogenous gene may be maintained in a cell as an insertion into the genome (nuclear or plastid) or as an episomal molecule.

The term “expression” refers to the amount of a nucleic acid or amino acid sequence (e.g., peptide, polypeptide, or protein) in a cell. The increased expression of a gene refers to the increased transcription of that gene. The increased expression of an amino acid sequence, peptide, polypeptide, or protein refers to the increased translation of a nucleic acid encoding the amino acid sequence, peptide, polypeptide, or protein.

The term “gene,” as used herein, may encompass genomic sequences that contain exons, particularly polynucleotide sequences encoding polypeptide sequences involved in a specific activity. The term further encompasses synthetic nucleic acids that did not derive from genomic sequence. In certain embodiments, the genes lack introns, as they are synthesized based on the known DNA sequence of cDNA and protein sequence. In other embodiments, the genes are synthesized, non-native cDNA wherein the codons have been optimized for expression in Y. lipolytica based on codon usage. The term can further include nucleic acid molecules comprising upstream, downstream, and/or intron nucleotide sequences.

The term “genetic modification” refers to the result of a transformation. Every transformation causes a genetic modification by definition.

The term “homolog”, as used herein, refers to (a) peptides, oligopeptides, polypeptides, proteins, and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived, and (b) nucleic acids which encode peptides, oligopeptides, polypeptides, proteins, and enzymes with the same characteristics described in (a).

“Inducible promoter” is a promoter that mediates the transcription of an operably linked gene in response to a particular stimulus.

The term “integrated” refers to a nucleic acid that is maintained in a cell as an insertion into the cell's genome, such as insertion into a chromosome, including insertions into a plastid genome.

“In operable linkage” refers to a functional linkage between two nucleic acid sequences, such a control sequence (typically a promoter) and the linked sequence (typically a sequence that encodes a protein, also called a coding sequence). A promoter is in operable linkage with a gene if it can mediate transcription of the gene.

The term “knockout mutation” or “knockout” refers to a genetic modification that prevents a native gene from being transcribed and translated into a functional protein.

The term “native” refers to the composition of a cell or parent cell prior to a transformation event. A “native gene” refers to a nucleotide sequence that encodes a protein that has not been introduced into a cell by a transformation event. A “native protein” refers to an amino acid sequence that is encoded by a native gene.

The terms “nucleic acid” refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.

The term “parent cell” refers to every cell from which a cell descended. The genome of a cell is comprised of the parent cell's genome and any subsequent genetic modifications to the parent cell's genome.

As used herein, the term “plasmid” refers to a circular DNA molecule that is physically separate from an organism's genomic DNA. Plasmids may be linearized before being introduced into a host cell (referred to herein as a linearized plasmid). Linearized plasmids may not be self-replicating, but may integrate into and be replicated with the genomic DNA of an organism.

The term “portion” refers to peptides, oligopeptides, polypeptides, protein domains, and proteins. A nucleotide sequence encoding a “portion of a protein” includes both nucleotide sequences that can be transcribed and/or translated and nucleotide sequences that must undergo one or more recombination events to be transcribed and/or translated. For example, a nucleic acid may comprise a nucleotide sequence encoding one or more amino acids of a selectable marker protein. This nucleic acid can be engineered to recombine with one or more different nucleotide sequences that encode the remaining portion of the protein. Such nucleic acids are useful for generating knockout mutations because only recombination with the target sequence is likely to reconstitute the full-length selectable marker gene whereas random-integration events are unlikely to result in a nucleotide sequence that can produce a functional marker protein. A “biologically-active portion” of a polypeptide is any amino acid sequence found in the polypeptide's amino acid sequence that is less than the full amino acid sequence but can perform the same function as the full-length polypeptide. A biologically-active portion of a diacylglycerol acyltransferase includes any amino acid sequence found in a full-length diacylglycerol acyltransferase that can catalyze the formation of triacylglycerol from diacylglycerol and acyl-CoA. A biologically-active portion of a polypeptide includes portions of the polypeptide that have the same activity as the full-length peptide and every portion that has more activity than background. For example, a biologically-active portion of a diacylglycerol acyltransferase may have 0.1, 0.5, 1, 2, 3, 4, 5, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.6, 99.7, 99.8, 99.9, 100, 100.1, 100.2, 100.3, 100.4, 100.5, 100.6, 100.7, 100.8, 100.9, 101, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 percent activity relative to the full-length polypeptide or higher. A biologically-active portion of a polypeptide may include portions of a peptide that lack a domain that targets the polypeptide to a cellular compartment.

A “promoter” is a nucleic acid control sequence that directs the transcription of a nucleic acid. As used herein, a promoter includes the necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.

“Recombinant” refers to a cell, nucleic acid, protein, or vector, which has been modified due to the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. Thus, e.g., recombinant cells can express genes that are not found within the native (non-recombinant) form of the cell or express native genes differently than those genes are expressed by a non-recombinant cell. Recombinant cells can, without limitation, include recombinant nucleic acids that encode for a gene product or for suppression elements such as mutations, knockouts, antisense, interfering RNA (RNAi), or dsRNA that reduce the levels of active gene product in a cell. A “recombinant nucleic acid” is a nucleic acid originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases, ligases, exonucleases, and endonucleases, or otherwise is in a form not normally found in nature. Recombinant nucleic acids may be produced, for example, to place two or more nucleic acids in operable linkage. Thus, an isolated nucleic acid or an expression vector formed in vitro by ligating DNA molecules that are not normally joined in nature, are both considered recombinant for the purposes of this invention. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it may replicate using the in vivo cellular machinery of the host cell; however, such nucleic acids, once produced recombinantly, although subsequently replicated intracellularly, are still considered recombinant for purposes of this invention. Similarly, a “recombinant protein” is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid.

The term “regulatory region” refers to nucleotide sequences that affect the transcription or translation of a gene but do not encode an amino acid sequence. Regulatory regions include promoters, operators, enhancers, and silencers.

“Transformation” refers to the transfer of a nucleic acid into a host organism or the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “recombinant”, “transgenic” or “transformed” organisms. Thus, isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. Typically, expression vectors include, for example, one or more cloned genes under the transcriptional control of 5′ and 3′ regulatory sequences and a selectable marker. Such vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or location-specific expression), a transcription initiation start site, a ribosome binding site, a transcription termination site, and/or a polyadenylation signal.

The term “transformed cell” refers to a cell that has undergone a transformation. Thus, a transformed cell comprises the parent's genome and an inheritable genetic modification.

The terms “triacylglyceride,” “triacylglycerol,” “triglyceride,” and “TAG” are esters comprised of glycerol and three fatty acids.

The term “triacylglycerol lipase” refers to any protein that can catalyze the removal of a fatty acid chain from a triacylglycerol. Triacylglycerol lipases include TGL3, TLG3/4, and TGL4.

The term “vector” refers to the means by which a nucleic acid can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include plasmids, linear DNA fragments, viruses, bacteriophage, pro-viruses, phagemids, transposons, and artificial chromosomes, and the like, that may or may not be able to replicate autonomously or integrate into a chromosome of a host cell.

Microbe Engineering

A. Overview

In certain embodiments of the invention, a microorganism is genetically modified to increase its triacylglycerol content.

Genes and gene products may be introduced into microbial host cells. Suitable host cells for expression of the genes and nucleic acid molecules are microbial hosts that can be found broadly within the fungal or bacterial families. Examples of suitable host strains include but are not limited to fungal or yeast species, such as Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Hansenula, Kluyveromyces, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Yarrowia, or bacterial species, such as members of proteobacteria and actinomycetes, as well as the genera Acinetobacter, Arthrobacter, Brevibacterium, Acidovorax, Bacillus, Clostridia, Streptomyces, Escherichia, Salmonella, Pseudomonas, and Cornyebacterium. Yarrowia lipolytica and Arxula adeninivorans are suited for use as a host microorganism because they can accumulate a large percentage of their weight as triacylglycerols.

Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are known to those skilled in the art. Any of these could be used to construct chimeric genes to produce any one of the gene products of the instant sequences. These chimeric genes could then be introduced into appropriate microorganisms via transformation techniques to provide high-level expression of the enzymes.

For example, a gene encoding an enzyme can be cloned in a suitable plasmid, and an aforementioned starting parent strain as a host can be transformed with the resulting plasmid. This approach can increase the copy number of each of the genes encoding the enzymes and, as a result, the activities of the enzymes can be increased. The plasmid is not particularly limited so long as it renders a desired genetic modification inheritable to the microorganism's progeny.

Vectors or cassettes useful for the transformation of suitable host cells are well known in the art. Typically the vector or cassette contains sequences that direct the transcription and translation of the relevant gene, a selectable marker, and sequences that allow autonomous replication or chromosomal integration. Suitable vectors comprise a region 5′ of the gene harboring transcriptional initiation controls and a region 3′ of the DNA fragment which controls transcriptional termination. It is preferred when both control regions are derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a production host.

Promoters, cDNAs, and 3′UTRs, as well as other elements of the vectors, can be generated through cloning techniques using fragments isolated from native sources (Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012); U.S. Pat. No. 4,683,202; incorporated by reference). Alternatively, elements can be generated synthetically using known methods (Gene 164:49-53 (1995)).

B. Homologous Recombination

Homologous recombination is the ability of complementary DNA sequences to align and exchange regions of homology. Transgenic DNA (“donor”) containing sequences homologous to the genomic sequences being targeted (“template”) is introduced into the organism and then undergoes recombination into the genome at the site of the corresponding homologous genomic sequences.

The ability to carry out homologous recombination in a host organism has many practical implications for what can be carried out at the molecular genetic level and is useful in the generation of a microbe that can produce a desired product. By its nature, homologous recombination is a precise gene targeting event and, hence, most transgenic lines generated with the same targeting sequence will be essentially identical in terms of phenotype, necessitating the screening of far fewer transformation events. Homologous recombination also targets gene insertion events into the host chromosome, potentially resulting in excellent genetic stability, even in the absence of genetic selection. Because different chromosomal loci will likely impact gene expression, even from exogenous promoters/UTRs, homologous recombination can be a method of querying loci in an unfamiliar genome environment and to assess the impact of these environments on gene expression.

A particularly useful genetic engineering approach using homologous recombination is to co-opt specific host regulatory elements such as promoters/UTRs to drive heterologous gene expression in a highly specific fashion.

Because homologous recombination is a precise gene targeting event, it can be used to precisely modify any nucleotide(s) within a gene or region of interest, so long as sufficient flanking regions have been identified. Therefore, homologous recombination can be used as a means to modify regulatory sequences impacting gene expression of RNA and/or proteins. It can also be used to modify protein coding regions in an effort to modify enzyme activities such as substrate specificity, affinities and Km, thereby affecting a desired change in the metabolism of the host cell. Homologous recombination provides a powerful means to manipulate the host genome resulting in gene targeting, gene conversion, gene deletion, gene duplication, gene inversion and exchanging gene expression regulatory elements such as promoters, enhancers and 3′UTRs.

Homologous recombination can be achieved by using targeting constructs containing pieces of endogenous sequences to “target” the gene or region of interest within the endogenous host cell genome. Such targeting sequences can either be located 5′ of the gene or region of interest, 3′ of the gene/region of interest or even flank the gene/region of interest. Such targeting constructs can be transformed into the host cell either as a supercoiled plasmid DNA with additional vector backbone, a PCR product with no vector backbone, or as a linearized molecule. In some cases, it may be advantageous to first expose the homologous sequences within the transgenic DNA (donor DNA) by cutting the transgenic DNA with a restriction enzyme. This step can increase the recombination efficiency and decrease the occurrence of undesired events. Other methods of increasing recombination efficiency include using PCR to generate transforming transgenic DNA containing linear ends homologous to the genomic sequences being targeted.

C. Vectors and Vector Components

Vectors for transforming microorganisms in accordance with the present invention can be prepared by known techniques familiar to those skilled in the art in view of the disclosure herein. A vector typically contains one or more genes, in which each gene codes for the expression of a desired product (the gene product) and is operably linked to one or more control sequences that regulate gene expression or target the gene product to a particular location in the recombinant cell.

1. Control Sequences

Control sequences are nucleic acids that regulate the expression of a coding sequence or direct a gene product to a particular location in or outside a cell. Control sequences that regulate expression include, for example, promoters that regulate transcription of a coding sequence and terminators that terminate transcription of a coding sequence. Another control sequence is a 3′ untranslated sequence located at the end of a coding sequence that encodes a polyadenylation signal. Control sequences that direct gene products to particular locations include those that encode signal peptides, which direct the protein to which they are attached to a particular location inside or outside the cell.

Thus, an exemplary vector design for expression of a gene in a microbe contains a coding sequence for a desired gene product (for example, a selectable marker, or an enzyme) in operable linkage with a promoter active in yeast. Alternatively, if the vector does not contain a promoter in operable linkage with the coding sequence of interest, the coding sequence can be transformed into the cells such that it becomes operably linked to an endogenous promoter at the point of vector integration.

The promoter used to express a gene can be the promoter naturally linked to that gene or a different promoter.

A promoter can generally be characterized as constitutive or inducible. Constitutive promoters are generally active or function to drive expression at all times (or at certain times in the cell life cycle) at the same level. Inducible promoters, conversely, are active (or rendered inactive) or are significantly up- or down-regulated only in response to a stimulus. Both types of promoters find application in the methods of the invention. Inducible promoters useful in the invention include those that mediate transcription of an operably linked gene in response to a stimulus, such as an exogenously provided small molecule, temperature (heat or cold), lack of nitrogen in culture media, etc. Suitable promoters can activate transcription of an essentially silent gene or upregulate, preferably substantially, transcription of an operably linked gene that is transcribed at a low level.

Inclusion of termination region control sequence is optional, and if employed, then the choice is primarily one of convenience, as the termination region is relatively interchangeable. The termination region may be native to the transcriptional initiation region (the promoter), may be native to the DNA sequence of interest, or may be obtainable from another source (See, e.g., Chen & Orozco, Nucleic Acids Research 16:8411 (1988)).

2. Genes and Codon Optimization

Typically, a gene includes a promoter, a coding sequence, and termination control sequences. When assembled by recombinant DNA technology, a gene may be termed an expression cassette and may be flanked by restriction sites for convenient insertion into a vector that is used to introduce the recombinant gene into a host cell. The expression cassette can be flanked by DNA sequences from the genome or other nucleic acid target to facilitate stable integration of the expression cassette into the genome by homologous recombination. Alternatively, the vector and its expression cassette may remain unintegrated (e.g., an episome), in which case, the vector typically includes an origin of replication, which is capable of providing for replication of the vector DNA.

A common gene present on a vector is a gene that codes for a protein, the expression of which allows the recombinant cell containing the protein to be differentiated from cells that do not express the protein. Such a gene, and its corresponding gene product, is called a selectable marker or selection marker. Any of a wide variety of selectable markers can be employed in a transgene construct useful for transforming the organisms of the invention.

For optimal expression of a recombinant protein, it is beneficial to employ coding sequences that produce mRNA with codons optimally used by the host cell to be transformed. Thus, proper expression of transgenes can require that the codon usage of the transgene matches the specific codon bias of the organism in which the transgene is being expressed. The precise mechanisms underlying this effect are many, but include the proper balancing of available aminoacylated tRNA pools with proteins being synthesized in the cell, coupled with more efficient translation of the transgenic messenger RNA (mRNA) when this need is met. When codon usage in the transgene is not optimized, available tRNA pools are not sufficient to allow for efficient translation of the transgenic mRNA resulting in ribosomal stalling and termination and possible instability of the transgenic mRNA.

D. Transformation

Cells can be transformed by any suitable technique including, e.g., biolistics, electroporation, glass bead transformation, and silicon carbide whisker transformation. Any convenient technique for introducing a transgene into a microorganism can be employed in the present invention. Transformation can be achieved by, for example, the method of D. M. Morrison (Methods in Enzymology 68:326 (1979)), the method by increasing permeability of recipient cells for DNA with calcium chloride (Mandel & Higa, J. Molecular Biology, 53:159 (1970)), or the like.

Examples of expression of transgenes in oleaginous yeast (e.g., Yarrowia lipolytica) can be found in the literature (Bordes et al., J. Microbiological Methods, 70:493 (2007); Chen et al., Applied Microbiology & Biotechnology 48:232 (1997)). Examples of expression of exogenous genes in bacteria such as E. coli are well known (Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012)).

Vectors for transformation of microorganisms in accordance with the present invention can be prepared by known techniques familiar to those skilled in the art. In one embodiment, an exemplary vector design for expression of a gene in a microorganism contains a gene encoding an enzyme in operable linkage with a promoter active in the microorganism. Alternatively, if the vector does not contain a promoter in operable linkage with the gene of interest, the gene can be transformed into the cells such that it becomes operably linked to a native promoter at the point of vector integration. The vector can also contain a second gene that encodes a protein. Optionally, one or both gene(s) is/are followed by a 3′ untranslated sequence containing a polyadenylation signal. Expression cassettes encoding the two genes can be physically linked in the vector or on separate vectors. Co-transformation of microbes can also be used, in which distinct vector molecules are simultaneously used to transform cells (Protist 155:381-93 (2004)). The transformed cells can be optionally selected based upon the ability to grow in the presence of the antibiotic or other selectable marker under conditions in which cells lacking the resistance cassette would not grow.

Exemplary Nucleic Acids, Cells, and Methods

A. Diacylglycerol Acyltransferase Nucleic Acid Molecules and Vectors

The diacylglycerol acyltransferase may be a type 1 diacylglycerol acyltransferase, type 2 diacylglycerol acyltransferase, type 3 diacylglycerol acyltransferase, or any other protein that catalyzes the conversion of diacylglycerol into a triacylglyceride. In some embodiments, the diacylglycerol acyltransferase is DGA1. For example, the diacylglycerol acyltransferase may be a DGA1 protein encoded by a DGAT2 gene selected from the group consisting of Arxula adeninivorans, Aspergillus terreus, Aurantiochytrium limacinum, Claviceps purpurea, Gloeophyllum trabeum, Lipomyces starkeyi, Microbotryum violaceum, Pichia guilliermondii, Phaeodactylum tricornutum, Puccinia graminis, Rhodosporidium diobovatum, Rhodosporidium toruloides, Rhodotorula graminis, and Yarrowia lipolytica.

The DGAT2 gene may have a nucleotide sequence set forth in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 62. In other embodiments, the DGAT2 gene is substantially identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 62, and the nucleotide sequence encodes a protein that retains the functional activity of a protein encoded by SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 62, yet differs in nucleotide sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGAT2 gene comprises an nucleotide sequence at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, or 62.

The DGA1 may have an amino acid sequence set forth in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61. In other embodiments, the DGA1 is substantially identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61, and retains the functional activity of the protein of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61, yet differs in amino acid sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGA1 protein comprises an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, or 61.

In some embodiments, the diacylglycerol acyltransferase is DGA2. For example, the diacylglycerol acyltransferase may be a DGA2 protein encoded by a DGAT1 gene found in an organism selected from the group consisting of Arxula adeninivorans, Aspergillus terreus, Chaetomium globosum, Claviceps purpurea, Lipomyces starkeyi, Metarhizium acridum, Ophiocordyceps sinensis, Phaeodactylum tricornutum, Pichia guilliermondii, Rhodosporidium toruloides, Rhodotorula graminis, Trichoderma virens, and Yarrowia lipolytica.

The DGAT1 gene may have a nucleotide sequence set forth in SEQ ID NO: 30, 32, 34, 36, 38, 40, 64, 66, 68, 70, 72, 74, or 76. In other embodiments, the DGAT1 gene is substantially identical to SEQ ID NO: 30, 32, 34, 36, 38, 40, 64, 66, 68, 70, 72, 74, or 76, and the nucleotide sequence encodes a protein that retains the functional activity of a protein encoded by SEQ ID NO: 30, 32, 34, 36, 38, 40, 64, 66, 68, 70, 72, 74, or 76, yet differs in nucleotide sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGAT1 gene comprises an nucleotide sequence at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 30, 32, 34, 36, 38, 40, 64, 66, 68, 70, 72, 74, or 76.

The DGA2 may have an amino acid sequence set forth in SEQ ID NO: 29, 31, 33, 35, 37, 39, 63, 65, 67, 69, 71, 73, or 75. In other embodiments, the DGA2 is substantially identical to SEQ ID NO: 29, 31, 33, 35, 37, 39, 63, 65, 67, 69, 71, 73, or 75, and retains the functional activity of the protein of SEQ ID NO: 29, 31, 33, 35, 37, 39, 63, 65, 67, 69, 71, 73, or 75, yet differs in amino acid sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGA2 protein comprises an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 29, 31, 33, 35, 37, 39, 63, 65, 67, 69, 71, 73, or 75.

In some embodiments, the diacylglycerol acyltransferase is DGA3. For example, the diacylglycerol acyltransferase may be a DGA3 protein encoded by a DGAT3 gene found in an organism selected from the group consisting of Ricinus communis and Arachis hypogaea.

The DGAT3 gene may have a nucleotide sequence set forth in SEQ ID NO: 84 or 86. In other embodiments, the DGAT3 gene is substantially identical to SEQ ID NO: 84 or 86, and the nucleotide sequence encodes a protein that retains the functional activity of a protein encoded by SEQ ID NO: 84 or 86, yet differs in nucleotide sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGAT3 gene comprises an nucleotide sequence at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 84 or 86.

The DGA3 may have an amino acid sequence set forth in SEQ ID NO: 83 or 85. In other embodiments, the DGA3 is substantially identical to SEQ ID NO: 83 or 85, and retains the functional activity of the protein of SEQ ID NO: 83 or 85, yet differs in amino acid sequence due to natural allelic variation or mutagenesis. In another embodiment, the DGA3 protein comprises an amino acid sequence at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more identical to SEQ ID NO: 83 or 85.

The DGAT1, DGAT2, and DGAT3 genes may comprise conservative substitutions, deletions, and/or insertions while still encoding a protein that has functional diacylglycerol acyltransferase activity. For example, the DGAT1, DGAT2, or DGAT3 codons may be optimized for a particular host cell, different codons may be substituted for convenience, such as to introduce a restriction site or to create optimal PCR primers, or codons may be substituted for another purpose. Similarly, the nucleotide sequence may be altered to create conservative amino acid substitutions, deletions, and/or insertions.

The DGA1, DGA2, and DGA3 polypeptides may comprise conservative substitutions, deletions, and/or insertions while still maintaining functional diacylglycerol acyltransferase activity. Conservative substitution tables are well known in the art (Creighton, Proteins (2d. ed., 1992)).

Amino acid substitutions, deletions and/or insertions may readily be made using recombinant DNA manipulation techniques. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. These methods include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), Quick Change Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis, and other site-directed mutagenesis protocols.

To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes can be at least 95% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions can then be compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Molecular Biology 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another embodiment, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller (Computer Applications in the Biosciences 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0 or 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, MEGABLAST, BLASTX, TBLASTN, TBLASTX, and BLASTP, and Clustal programs, e.g., ClustalW, ClustalX, and Clustal Omega.

Sequence searches are typically carried out using the BLASTN program, when evaluating a given nucleic acid sequence relative to nucleic acid sequences in the GenBank DNA Sequences and other public databases. The BLASTX program is effective for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases.

An alignment of selected sequences in order to determine “% identity” between two or more sequences is performed using for example, the CLUSTAL-W program.

A “coding sequence” or “coding region” refers to a nucleic acid molecule having sequence information necessary to produce a protein product, such as an amino acid or polypeptide, when the sequence is expressed. The coding sequence may comprise and/or consist of untranslated sequences (including introns or 5′ or 3′ untranslated regions) within translated regions, or may lack such intervening untranslated sequences (e.g., as in cDNA).

The abbreviation used throughout the specification to refer to nucleic acids comprising and/or consisting of nucleotide sequences are the conventional one-letter abbreviations. Thus when included in a nucleic acid, the naturally occurring encoding nucleotides are abbreviated as follows: adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Also, unless otherwise specified, the nucleic acid sequences presented herein is the 5′→3′direction.

As used herein, the term “complementary” and derivatives thereof are used in reference to pairing of nucleic acids by the well-known rules that A pairs with T or U and C pairs with G. Complement can be “partial” or “complete”. In partial complement, only some of the nucleic acid bases are matched according to the base pairing rules; while in complete or total complement, all the bases are matched according to the pairing rule. The degree of complement between the nucleic acid strands may have significant effects on the efficiency and strength of hybridization between nucleic acid strands as well known in the art. The efficiency and strength of said hybridization depends upon the detection method.

As used herein, “DGA1” means a diacylglycerol acyltransferase type 2 (DGAT2). DGA1 is an integral membrane protein that catalyzes the final enzymatic step in oil biosynthesis and the production of triacylglycerols in plants, fungi, and mammals. The DGA1 may play a key role in altering the quantity of long-chain polyunsaturated fatty acids produced in oils of oleaginous organisms. DGA1 is related to the acyl-coenzyme A:cholesterol acyltransferase (“ACAT”). This enzyme is responsible for transferring an acyl group from acyl-coenzyme-A to the sn-3 position of 1,2-diacylglycerol (“DAG”) to form triacylglycerol (“TAG”) (thereby involved in the terminal step of TAG biosynthesis). DGA1 is associated with membrane and lipid body fractions in plants and fungi, particularly, in oilseeds where it contributes to the storage of carbon used as energy reserves. TAG is believed to be an important chemical for storage of energy in cells. DGA1 is known to regulate TAG structure and direct TAG synthesis.

The DGA1 polynucleotide and polypeptide sequences may be derived from highly oleaginous organisms having very high, native levels of lipid accumulation. (Bioresource Technology 144:360-69 (2013); Progress Lipid Research 52:395-408 (2013); Applied Microbiology & Biotechnology 90:1219-27 (2011); European Journal Lipid Science & Technology 113:1031-51 (2011); Food Technology & Biotechnology 47:215-20 (2009); Advances Applied Microbiology 51:1-51 (2002); Lipids 11:837-44 (1976)). A list of organisms with a reported lipid content of about 50% and higher is shown in Table 1. R. toruloides and L. starkeyi have the highest lipid content. Among the organisms in Table 1, five have publicly accessible sequence for DGA1, R. toruloides, L. starkeyi, A. limacinum, A. terreus, and C. purpurea (bolded in Table 1).

TABLE 1 List of oleaginous fungi with reported lipid contents of about 50% and above. Organisms with publicly accessible sequences for a DGA1 gene are in bold. Fungi with reported high lipid content

 

 

 

Cryptococcus albidus Cryptococcus curvatus Cryptococcus ramirezgomezianus Cryptococcus terreus Cryptococcus wieringae Cunninghamella echinulata Cunninghamella japonica Leucosporidiella creatinivora Lipomyces lipofer

 

Lipomyces tetrasporus Mortierella isabellina Prototheca zopfii Rhizopus arrhizus Rhodosporidium babjevae Rhodosporidium paludigenum

 

Rhodotorula glutinis Rhodotorula mucilaginosa Tremella enchepala Trichosporon cutaneum Trichosporon fermentans

Nucleic acid constructs for overexpressing the DGA1 gene were described in U.S. Ser. No. 61/943,664 (hereby incorporated by reference). FIG. 1 shows expression construct pNC243 used for overexpression of the R. toruloides DGA1 gene NG66 (SEQ ID No. 6) in Y. lipolytica. DGA1 expression constructs were linearized before transformation by PacI/NotI restriction digest (FIG. 1). The linear expression constructs each included expression cassette for DGA1 gene and for Nat1 gene, used as marker for selection with nourseothricin (NAT).

Nucleic acid constructs for overexpressing the DGA2 gene and/or other diacylglycerol acyltransferases may be created using the methods described above and/or other methods known in the art.

B. Triacylglycerol Lipase Nucleic Acid Molecules and Vectors

Triacylglycerol lipase depletes a cell's triacylglycerol by removing one or more fatty acid chains. Thus, decreasing the net triacylglycerol lipase activity of a cell may increase the cell's triacylglycerol. This decrease may be accomplished by reducing the efficiency of the enzyme, e.g., by mutating amino acids in its active site, or by reducing the expression of the enzyme. For example, a TGL3 knockout mutation will decrease the activity of a triacylglycerol lipase because it prevents the cell from transcribing TGL3.

In some embodiments, the triacylglycerol lipase is TGL3, TGL3/4, or TGL4.

The TGL3 gene in Y. lipolytica encodes the triacylglycerol lipase protein TGL3 (SEQ ID No. 19). SEQ ID No. 20 contains the TGL3 nucleotide sequence, 100 upstream nucleotides, and 100 downstream. Thus, the SEQ ID No. 20 nucleotide sequence may be used to design a nucleic acid capable of recombining with a nucleic acid sequence in the native Y. lipolytica triacylglycerol lipase gene.

Knockout cassettes SEQ ID Nos. 27 and 28 are capable of recombining with the native TGL3 gene in Y. lipolytica. Thus, in some embodiments, the nucleic acids encoded by SEQ ID Nos. 27 and 28 may be used to generate a triacylglycerol lipase knockout mutation in Y. lipolytica. SEQ ID Nos. 27 and 28 each contain portions of a hygromycin resistance gene hph. Neither isolated sequence encodes a functional protein, but the two sequences are capable of encoding a functional kinase that confers hygromycin resistance upon successful recombination. Further, neither SEQ ID No. 27 nor SEQ ID No. 28 contains a promoter or terminator, and thus, they rely on homologous recombination with the Y. lipolytica TGL3 gene in order for the hph gene to be transcribed and translated. In this way, successfully transformed oleaginous cells may be selected by growing the cells on medium containing hygromycin.

Knockout cassette SEQ ID NO. 27 may be prepared by amplifying a hygromycin resistance gene hph (SEQ ID NO. 22) with primer NP1798 (SEQ ID NO. 23) and primer NP656 (SEQ ID NO. 24). Knockout cassette SEQ ID NO. 28 may be prepared by amplifying a hygromycin resistance gene hph (SEQ ID NO. 22) with primer NP655 (SEQ ID NO. 25) and primer NP1799 (SEQ ID NO. 26).

Different approaches may be used to design nucleic acids that knockout the TGL3 gene in Y. lipolytica (Biochimica Biophysica Acta 1831:1486-95 (2013)). The methods disclosed herein and other methods known in the art may be used to knockout triacylglycerol different lipase genes in other species. For example, these methods may be used to reduce the activity of the TGL3 gene of Arxula adeninivorans (SEQ ID NO:78), the TGL3/4 gene of Arxula adeninivorans (SEQ ID NO:80), and/or the TGL4 gene of Arxula adeninivorans (SEQ ID NO:82). Similarly, these methods may be used to reduce the activity of the TGL4 gene in Y. lipolytica (SEQ ID NO:88).

C. Transformed Oleaginous Cell

In some embodiments, the transformed oleaginous cell is a prokaryotic cell, such as a bacterial cell. In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell, a yeast cell, a filamentous fungi cell, a protist cell, an algae cell, an avian cell, a plant cell, or an insect cell.

The cell may be selected from the group consisting of Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, and Yarrowia.

In some embodiments, the cell is selected from the group of consisting of Arxula adeninivorans, Aspergillus niger, Aspergillus orzyae, Aspergillus terreus, Aurantiochytrium limacinum, Candida utilis, Claviceps purpurea, Cryptococcus albidus, Cryptococcus curvatus, Cryptococcus ramirezgomezianus, Cryptococcus terreus, Cryptococcus wieringae, Cunninghamella echinulata, Cunninghamella japonica, Geotrichum fermentans, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Kodamaea ohmeri, Leucosporidiella creatinivora, Lipomyces lipofer, Lipomyces starkeyi, Lipomyces tetrasporus, Mortierella isabellina, Mortierella alpina, Ogataea polymorpha, Pichia ciferrii, Pichia guilliermondii, Pichia pastoris, Pichia stipites, Prototheca zopfii, Rhizopus arrhizus, Rhodosporidium babjevae, Rhodosporidium toruloides, Rhodosporidium paludigenum, Rhodotorula glutinis, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Tremella enchepala, Trichosporon cutaneum, Trichosporon fermentans, Wickerhamomyces ciferrii, and Yarrowia lipolytica.

In certain embodiments, the transformed oleaginous cell is a high-temperature tolerant yeast cell. In some embodiments the transformed oleaginous cell is Kluyveromyces marxianus.

In certain embodiments, the cell is Yarrowia lipolytica or Arxula adeninivorans.

D. Increasing Diacylglycerol Acyltransferase in a Cell

A protein's activity may be increased by overexpressing the protein. Proteins may be overexpressed in a cell using a variety of genetic modifications. In some embodiments, the genetic modification increases the expression of a native diacylglycerol acyltransferase. A native diacylglycerol acyltransferase may be overexpressed by modifying the upstream transcription regulators of a native diacylglycerol acyltransferase gene, for example, by increasing the expression of a transcription activator or decreasing the expression of a transcription repressor. Alternatively, the promoter of a native diacylglycerol acyltransferase gene may be substituted with a constitutively active or inducible promoter by recombination with an exogenous nucleic acid.

In some embodiments, the genetic modification encodes at least one copy of a diacylglycerol acyltransferase gene. The diacylglycerol acyltransferase gene may be a gene native to the cell or from a different species. In certain embodiments, the diacylglycerol acyltransferase gene is the type II diacylglycerol acyltransferase gene from Arxula adeninivorans, Aspergillus terreus, Aurantiochytrium limacinum, Claviceps purpurea, Gloeophyllum trabeum, Lipomyces starkeyi, Microbotryum violaceum, Pichia guilliermondii, Phaeodactylum tricornutum, Puccinia graminis, Rhodosporidium diobovatum, Rhodosporidium toruloides, Rhodotorula graminis, or Yarrowia lipolytica. In certain embodiments, the diacylglycerol acyltransferase gene is the type I diacylglycerol acyltransferase gene from Arxula adeninivorans, Aspergillus terreus, Chaetomium globosum, Claviceps purpurea, Lipomyces starkeyi, Metarhizium acridum, Ophiocordyceps sinensis, Phaeodactylum tricornutum, Pichia guilliermondii, Rhodosporidium toruloides, Rhodotorula graminis, Trichoderma virens, or Yarrowia lipolytica. In certain embodiments, the diacylglycerol acyltransferase gene is the type III diacylglycerol acyltransferase gene from Ricinus communis or Arachis hypogaea. In certain embodiments, diacylglycerol acyltransferase is overexpressed by transforming a cell with a gene encoding a diacylglycerol acyltransferase gene. The genetic modification may encode one or more than one copy of a diacylglycerol acyltransferase gene. In certain embodiments, the genetic modification encodes at least one copy of the DGA1 protein from R. toruloides. In some embodiments, the genetic modification encodes at least one copy of the DGA1 protein from R. toruloides and the transformed oleaginous cell is Y. lipolytica.

In certain embodiments, the diacylglycerol acyltransferase gene is inheritable to the progeny of a transformed oleaginous cell. In some embodiments, the diacylglycerol acyltransferase gene is inheritable because it resides on a plasmid. In certain embodiments, the diacylglycerol acyltransferase gene is inheritable because it is integrated into the genome of the transformed oleaginous cell.

E. Decreasing Triacylglycerol Lipase Activity in a Cell

In some embodiments, the transformed oleaginous cell comprises a genetic modification that decreases the activity of a native triacylglycerol lipase. Such genetic modifications may affect a protein that regulates the transcription of a triacylglycerol lipase gene, including modifications that decrease the expression of a transcription activator and/or increase the expression of a transcription repressor. Modifications that affect a regulator protein may both decrease the expression of triacylglycerol lipase and alter other gene expression profiles that shift the cellular equilibrium toward increased triacylglycerol accumulation. Alternatively, the genetic modification may be the introduction of a small interfering RNA, or a nucleic acid that encodes a small interfering RNA. In other embodiments, the genetic modification consists of the homologous recombination of a nucleic acid and the regulatory region of a native triacylglycerol lipase gene, including an operator, promoter, sequences upstream from the promoter, enhancers, and sequences downstream of the gene.

In some embodiments the transformed oleaginous cell comprises a genetic modification consisting of a homologous recombination event. In certain embodiments, the transformed oleaginous cell comprises a genetic modification consisting of a homologous recombination event between a native triacylglycerol lipase gene and a nucleic acid. Thus, the genetic modification deletes the triacylglycerol lipase gene, prevents its transcription, or prevents the transcription of a gene that can be transcribed into a fully-active protein. A homologous recombination event may mutate or delete a portion of a native triacylglycerol lipase gene. For example, the homologous recombination event may mutate one or more residues in the active site of a native triacylglycerol lipase, thereby reducing the efficiency of the lipase or rendering it inactive. Alternatively, the homologous recombination event may affect post-translational modification, folding, stability, or localization within the cell. In certain embodiments, the genetic modification is a triacylglycerol lipase knockout mutation. Knockout mutations are preferable because they eliminate a pathway that depletes a cell's triacylglycerol content, thereby increasing the triacylglycerol content of a cell.

A knockout mutation may delete the triacylglycerol lipase gene. Additionally, the knockout mutation may substitute the triacylglycerol lipase gene with a gene that encodes a different protein. The gene may be operably linked to an exogenous promoter. In certain embodiments, the gene is not linked to an exogenous promoter, and instead, the gene is configured to recombine with the triacylglycerol lipase gene such that the triacylglycerol lipase gene's promoter drives transcription of the gene. Thus, the gene is less likely to be expressed if it randomly integrates into the cell's genome. Methods for creating knockouts are well-known in the art (See, e.g., Fickers et al., J. Microbiological Methods 55:727 (2003)).

In certain embodiments, the genetic modification comprises two homologous recombination events. In the first event, a nucleic acid encoding a portion of a gene recombines with the triacylglycerol lipase gene, and in the second event, a nucleic acid encoding the remaining portion of the gene recombines with the triacylglycerol lipase gene. The two portions of the gene are designed such that neither portion is functional unless they recombine with each other. These two events further reduce the likelihood that the gene can be expressed following random integration events.

In certain embodiments, the gene encodes a dominant selectable marker. Thus, knockout cells may be selected by screening for the marker. In some embodiments, the dominant selectable marker is a drug resistance marker. A drug resistance marker is a dominant selectable marker that, when expressed by a cell, allows the cell to grow and/or survive in the presence of a drug that would normally inhibit cellular growth and/or survival. Cells expressing a drug resistance marker can be selected by growing the cells in the presence of the drug. In some embodiments, the drug resistance marker is an antibiotic resistance marker. In some embodiments, the drug resistance marker confers resistance to a drug selected from the group consisting of Amphotericin B, Candicidin, Filipin, Hamycin, Natamycin, Nystatin, Rimocidin, Bifonazole, Butoconazole, Clotrimazole, Econazole, Fenticonazole, Isoconazole, Ketoconazole, Luliconazole, Miconazole, Omoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Albaconazole, Fluconazole, Isavuconazole, Itraconazole, Posaconazole, Ravuconazole, Terconazole, Voriconazole, Abafungin, Amorolfin, Butenafine, Naftifine, Terbinafine, Anidulafungin, Caspofungin, Micafungin, Benzoic acid, Ciclopirox, Flucytosine, 5-fluorocytosine, Griseofulvin, Haloprogin, Polygodial, Tolnaftate, Crystal violet, Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, Spectinomycin, Geldanamycin, Herbimycin, Rifaximin, Streptomycin, Loracarbef, Ertapenem, Doripenem, Imipenem, Meropenem, Cefadroxil, Cefazolin, Cefalotin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, Ceftobiprole, Teicoplanin, Vancomycin, Telavancin, Clindamycin, Lincomycin, Daptomycin, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, Spiramycin, Aztreonam, Furazolidone, Nitrofurantoin, Linezolid, Posizolid, Radezolid, Torezolid, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cloxacillin, Dicloxacillin, Flucloxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Penicillin G, Temocillin, Ticarcillin, clavulanate, sulbactam, tazobactam, clavulanate, Bacitracin, Colistin, Polymyxin B, Ciprofloxacin, Enoxacin, Gatifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, Temafloxacin, Mafenide, Sulfacetamide, Sulfadiazine, Silver sulfadiazine, Sulfadimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, Co-trimoxazole, Sulfonamidochrysoidine, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, Tetracycline, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinamide, Rifampicin, Rifabutin, Rifapentine, Streptomycin, Arsphenamine, Chloramphenicol, Fosfomycin, Fusidic acid, Metronidazole, Mupirocin, Platensimycin, Quinupristin, Dalfopristin, Thiamphenicol, Tigecycline, Tinidazole, Trimethoprim, Geneticin, Nourseothricin, Hygromycin, Bleomycin, and Puromycin.

In some embodiments, the dominant selectable marker is a nutritional marker. A nutritional marker is a dominant selectable marker that, when expressed by the cell, enables the cell to grow or survive using one or more particular nutrient sources. Cells expressing a nutritional marker can be selected by growing the cells under limiting nutrient conditions in which cells expressing the nutritional marker can survive and/or grow, but cells lacking the nutrient marker cannot. In some embodiments, the nutritional marker is selected from the group consisting of Orotidine 5-phosphate decarboxylase, Phosphite specific oxidoreductase, Alpha-ketoglutarate-dependent hypophosphite dioxygenase, Alkaline phosphatase, Cyanamide hydratase, Melamine deaminase, Cyanurate amidohydrolase, Biuret hydrolyase, Urea amidolyase, Ammelide aminohydrolase, Guanine deaminase, Phosphodiesterase, Phosphotriesterase, Phosphite hydrogenase, Glycerophosphodiesterase, Parathion hydrolyase, Phosphite dehydrogenase, Dibenzothiophene desulfurization enzyme, Aromatic desulfinase, NADH-dependent FMN reductase, Aminopurine transporter, Hydroxylamine oxidoreductase, Invertase, Beta-glucosidase, Alpha-glucosidase, Beta-galactosidase, Alpha-galactosidase, Amylase, Cellulase, and Pullulonase.

Different approaches may be used to knockout the TGL3 gene in Y. lipolytica (See, e.g., Dulermo et al., Biochimica Biophysica Acta 1831:1486 (2013)). The methods disclosed herein and other methods known in the art may be used to knockout different triacylglycerol lipase genes in other species. For example, these methods may be used to knockout the TGL3 gene of Arxula adeninivorans (SEQ ID NO:78), the TGL3/4 gene of Arxula adeninivorans (SEQ ID NO:80), or the TGL4 gene of Arxula adeninivorans (SEQ ID NO:82). Similarly, these methods may be used to knockout the TGL4 gene of Y. lipolytica (SEQ ID NO:88).

In some embodiments, a genetic modification decreases the expression of a native triacylglycerol lipase gene by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100 percent.

In some embodiments, a genetic modification decreases the efficiency of a native triacylglycerol lipase gene by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100 percent.

In some embodiments, a genetic modification decreases the activity of a native triacylglycerol lipase gene by 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100 percent.

F. Decreasing Triacylglycerol Lipase Activity in a Cell with Concomitant Overexpression of Diacylglycerol Acyltransferase

In some embodiments, the transformed oleaginous cell comprises a triacylglycerol lipase knockout mutation and a genetic modification that increase the expression of a native diacylglycerol acyltransferase. In certain embodiments, the transformed oleaginous cell comprises a triacylglycerol lipase knockout mutation and a genetic modification that encodes at least one copy of a diacylglycerol acyltransferase gene that is either native to the cell or from a different species of cell. In other embodiments, a TGL3 gene is disrupted and a DGA1 protein is overexpressed.

In some embodiments, one nucleic acid increases the expression of a native diacylglycerol acyltransferase or encodes at least one copy of a diacylglycerol acyltransferase gene and a second nucleic acid decreases the activity of a triacylglycerol lipase in the cell. In some embodiments the same nucleic acid encodes at least one copy of a diacylglycerol acyltransferase gene and decreases the activity of a triacylglycerol lipase in the cell. For example, the nucleic acid designed to knock out a triacylglycerol lipase gene may also contain a copy of a diacylglycerol acyltransferase gene.

G. Triacylglycerol Production

In certain embodiments, the transformed oleaginous cell are grown in the presence of exogenous fatty acids, glucose, ethanol, xylose, sucrose, starch, starch dextrin, glycerol, cellulose, and/or acetic acid. These substrates may be added during cultivation to increase lipid production. The exogenous fatty acids may include stearate, oleic acid, linoleic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, α-linolenic acid, stearidonic acid, eicosatetraenoic acid, eicosapenteaenoic acid, docosapentaenoic acid, eicosadienoic acid, and/or eicosatrienoic acid.

In certain embodiments, the present invention relates to a product produced by a modified host cell described herein. In certain embodiments, the product is an oil, lipid, or triacylglycerol. In some embodiments, the product is palmitic acid, palmitoleic acid, stearic acid, oleic acid, or linoleic acid. In certain embodiments, the product is a saturated fatty acid. Thus, the product may be caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, or cerotic acid. In some embodiments, the product is an unsaturated fatty acid. Thus, the product may be myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapenteaenoic acid, erucic acid, or docosahexaenoic acid.

In some embodiments, the product comprises an 18-carbon fatty acid. In some embodiments, the product comprises oleic acid, stearic acid, or linoleic acid. For example, the product may be oleic acid.

In some embodiments, the present invention relates to a transformed oleaginous cell comprising a first genetic modification and a second genetic modification. The first genetic modification may increase the activity of a native diacylglycerol acyltransferase. Alternatively, the first genetic modification may encode at least one copy of a diacylglycerol acyltransferase gene native to the oleaginous cell or from a different species. The second genetic modification may decrease the activity of a triacylglycerol lipase in the oleaginous cell.

In some embodiments, the second genetic modification is a triacylglycerol lipase knockout mutation.

In some embodiments, the first genetic modification encodes at least one copy of a diacylglycerol acyltransferase gene native to the oleaginous cell or from a different species. In some embodiments, a diacylglycerol acyltransferase gene is integrated into the genome of the cell.

In some embodiments, the transformed oleaginous cell is selected from the group consisting of algae, bacteria, molds, fungi, plants, and yeasts. In certain embodiments, the transformed oleaginous cell is a yeast. The transformed oleaginous cell may be selected from the group consisting of Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, and Yarrowia. In certain embodiments, the transformed oleaginous cell is selected from the group consisting of Arxula adeninivorans, Aspergillus niger, Aspergillus orzyae, Aspergillus terreus, Aurantiochytrium limacinum, Candida utilis, Claviceps purpurea, Cryptococcus albidus, Cryptococcus curvatus, Cryptococcus ramirezgomezianus, Cryptococcus terreus, Cryptococcus wieringae, Cunninghamella echinulata, Cunninghamella japonica, Geotrichum fermentans, Hansenula polymorpha, Kluyveromyces lactis, Kluyveromyces marxianus, Kodamaea ohmeri, Leucosporidiella creatinivora, Lipomyces lipofer, Lipomyces starkeyi, Lipomyces tetrasporus, Mortierella isabellina, Mortierella alpina, Ogataea polymorpha, Pichia ciferrii, Pichia guilliermondii, Pichia pastoris, Pichia stipites, Prototheca zopfii, Rhizopus arrhizus, Rhodosporidium babjevae, Rhodosporidium toruloides, Rhodosporidium paludigenum, Rhodotorula glutinis, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Tremella enchepala, Trichosporon cutaneum, Trichosporon fermentans, Wickerhamomyces ciferrii, and Yarrowia lipolytica. In other embodiments, the transformed oleaginous cell is Yarrowia lipolytica or Arxula adeninivorans.

In some embodiments, the diacylglycerol acyltransferase gene is a type II diacylglycerol acyltransferase gene. The diacylglycerol acyltransferase gene may be a type II diacylglycerol acyltransferase gene from Arxula adeninivorans, Aspergillus terreus, Aurantiochytrium limacinum, Claviceps purpurea, Gloeophyllum trabeum, Lipomyces starkeyi, Microbotryum violaceum, Pichia guilliermondii, Phaeodactylum tricornutum, Puccinia graminis, Rhodosporidium diobovatum, Rhodosporidium toruloides, Rhodotorula graminis, or Yarrowia lipolytica.

In some embodiments, the diacylglycerol acyltransferase gene is a type I diacylglycerol acyltransferase gene. The diacylglycerol acyltransferase gene may be a type I diacylglycerol acyltransferase gene from Arxula adeninivorans, Aspergillus terreus, Chaetomium globosum, Claviceps purpurea, Lipomyces starkeyi, Metarhizium acridum, Ophiocordyceps sinensis, Phaeodactylum tricomuturn, Pichia guilliermondii, Rhodosporidium toruloides, Rhodotorula graminis, Trichoderma virens, or Yarrowia lipolytica.

In some embodiments, the diacylglycerol acyltransferase gene is a type III diacylglycerol acyltransferase gene. The diacylglycerol acyltransferase gene may be a type III diacylglycerol acyltransferase gene from Ricinus communis or Arachis hypogaea.

In some embodiments, the triacylglycerol lipase is TGL3, TGL3/4, or TGL4.

In some embodiments, the present invention relates to a product derived from a transformed oleaginous cell. In certain embodiments, the product is an oil, lipid, or triacylglycerol.

In some embodiments, the present invention relates to a method of increasing the lipid content of a cell, comprising transforming a parent cell with a first nucleic acid and a second nucleic acid. The first nucleic acid may increase the activity of a native diacylglycerol acyltransferase or comprise a diacylglycerol acyltransferase gene. The second nucleic acid may decrease the activity of a triacylglycerol lipase. The first nucleic acid and second nucleic acid may be the same. Thus, in some embodiments, the present invention relates to a method of increasing the lipid content of a cell comprising transforming a parent cell with a nucleic acid, wherein the nucleic acid decreases the activity of a triacylglycerol lipase in the cell and either comprises a diacylglycerol acyltransferase gene or increases the activity of a native diacylglycerol acyltransferase.

In some embodiments, the present invention relates to a method of increasing the triacylglycerol content of a cell, comprising providing a cell and growing the cell. The cell may comprise a first genetic modification and a second genetic modification. The first genetic modification may increase the activity of a native diacylglycerol acyltransferase or encode at least one copy of a diacylglycerol acyltransferase gene either native to the oleaginous cell or from a different species. The second genetic modification may decrease the activity of a triacylglycerol lipase in the cell. The cell may be grown under conditions whereby the first genetic modification is expressed, thereby producing a triacylglycerol. In some embodiments, the triacylglycerol is recovered.

In some embodiments, the first nucleic acid comprises a diacylglycerol acyltransferase gene. The diacylglycerol acyltransferase gene may encode a type I diacylglycerol acyltransferase polypeptide, a type II diacylglycerol acyltransferase polypeptide, or a type III diacylglycerol acyltransferase polypeptide.

In some embodiments, the diacylglycerol acyltransferase gene comprises a nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 84, or SEQ ID NO: 86, or a complement of any one of them.

In some embodiments, the diacylglycerol acyltransferase gene comprises a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleotide sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 84, or SEQ ID NO: 86, or a complement of any one of them.

In some embodiments, the diacylglycerol acyltransferase polypeptide comprises an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61; SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 85, or a biologically-active portion of any one of them.

In some embodiments, the diacylglycerol acyltransferase polypeptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to an amino acid sequence forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61; SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 85, or a biologically-active portion of any one of them.

In some embodiments, the second nucleic acid is capable of recombining with a nucleic acid sequence in a triacylglycerol lipase gene and/or a nucleic acid sequence in the regulatory region of a triacylglycerol lipase gene. In certain embodiments, the second nucleic acid is capable of recombining with a nucleic acid sequence in a TGL3, TGL3/4, or TGL4 gene and/or a nucleic acid sequence in the regulatory region of a TGL3, TGL3/4, or TGL4 gene. The second nucleic acid may comprise a gene encoding a protein or a portion of a protein. In certain embodiments, the second nucleic acid comprises a gene encoding a protein that confers resistance to a drug. In other embodiments, the second nucleic acid comprises a gene encoding a protein that enables the cell to grow or proliferate more quickly on one or more particular nutrient sources than a native organism of the same species.

The present description is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references (including literature references, issued patents, published patent applications and GenBank Accession numbers as cited throughout this application) are hereby expressly incorporated by reference. When definitions of terms in documents that are incorporated by reference herein conflict with those used herein, the definitions used herein govern.

EXEMPLIFICATION Example 1: Method to Overexpress DGA1 in Y. lipolytica

Nucleic acid constructs for overexpressing the DGA1 gene were described in U.S. Ser. No. 61/943,664 (hereby incorporated by reference). FIG. 1 shows expression construct pNC243 used for overexpression of the R. toruloides DGA1 gene NG66 (SEQ ID No. 6) in Y. lipolytica. DGA1 expression constructs were linearized before transformation by a PacI/NotI restriction digest. The linear expression constructs each included an expression cassette for the DGA1 gene and for the Nat1 gene, used as a marker for selection with nourseothricin (NAT).

DGA1 expression constructs were randomly integrated into the genome of E lipolytica strain NS18 (obtained from ARS Culture Collection, NRRL # YB 392) using a transformation protocol as described in Chen (Applied Microbiology & Biotechnology 48:232-35 (1997)). Transformants were selected on YPD plates with 500 μg/mL NAT and screened for the ability to accumulate lipids by a fluorescent staining lipid assay as described below. For each expression construct, eight transformants were analysed.

For most constructs, there was significant colony variation between the transformants, likely due to the lack of a functional DGA1 expression cassette in cells that only obtained a functional Nat1 cassette, or due to a negative effect of the site of DGA1 integration on DGA1 expression. Nevertheless, all transformants had a significant increase in lipid content.

Overexpression of native Y. lipolytica DGA1 (NG15) under a strong promoter increased lipid content as measured by cell fluorescence by about 2-fold compared to the parental strain NS18. Transformants that demonstrated the highest fluorescence (about 3-fold higher compared to NS18) were generated by the overexpression of R. toruloides DGA1 (NG66, NG67) and L. starkeyi DGA1 (NG68).

In certain experiments, the effect of native R. toruloides DGA1 (NG49) overexpression on lipid production in Y. lipolytica was not as high as the effect of synthetic versions of R. toruloides DGA1 genes that did not contain introns. This result may indicate that the gene splicing of the R. toruloides DGA1 gene in Y. lipolytica was not very efficient. In certain experiments, codon optimization of the R. toruloides DGA1 gene for expression in Y. lipolytica did not have a positive effect on lipid production.

In order to select strains with the highest lipid production level, Y. lipolytica strain NS18 transformants expressing NG15 (Y. lipolytica DGA1) or NG66 (R. toruloides DGA1) were screened. For NG15, about 50 colonies were screened by lipid assay for the highest lipid accumulation, and the best transformant was named NS249. For NG66, 80 colonies were screened and the 8 best colonies were selected for further analysis. Strain NS249 and the 8 selected NG66 transformants were grown in shake flasks and analysed by the lipid assay for lipid content and by HPLC for glucose consumption. Y. lipolytica strains overexpressing R. toruloides DGA1 have significantly higher lipid content than Y. lipolytica strains with native Y. lipolytica DGA1 gene expressed under the same promoter as R. toruloides DGA1. At the same time, NG66 transformants had significantly less glucose left in the media, demonstrating that NG66 was more efficient in converting glucose to lipids. The difference in efficiency between two DGA1 genes may be attributed to either a higher level of expression of R. toruloides DGA1 in Y. lipolytica or a higher level of R. toruloides DGA1 specific activity, or both. One of the best performing transformants with an integrated NG66 gene was named NS281.

Example 2: Method to Knockout Triacylglycerol Lipase Knockout Gene in Y. lipolytica

In order to test the idea that combining DGA1 overexpression with a TGL3 deletion leads to higher lipid accumulation in Y. lipolytica, we deleted TGL3 in Y. lipolytica wild-type strain NS18 (obtained from NRLL # YB-392) and its DGA1 overexpressing derivative NS281. NS281 overexpresses the DGA1 gene from Rhodosporidium toruloides as described above. The Y. lipolytica TGL3 gene (YALI0D17534g, SEQ ID NO: 20) was deleted as follows: A two-fragment deletion cassette was amplified by PCR from a plasmid containing the hygromycin resistance gene (“hph,” SEQ ID NO: 22) using primer pairs NP1798-NP656 and NP655-NP1799 (SEQ ID NOs: 23-26). The resulting PCR fragments (SEQ ID NOs: 27 & 28) were co-transformed into NS18 and NS281 according to the protocol developed in U.S. Ser. No. 61/819,746 (hereby incorporated by reference). The omission of a promoter and terminator in the hph cassette and the splitting of the hph coding sequence into two PCR fragments reduce the probability that random integration of these pieces will confer hygromycin resistance. The hph gene should only be expressed if it integrates at the TGL3 locus by homologous recombination so that the TGL3 promoter and terminator can direct its transcription. Hygromycin resistant colonies were screened by PCR to confirm the absence of TGL3 and the presence of a tgl3::hyg specific product. Deletion of TGL3 in NS18 resulted in strain NS421. Deletion of TGL3 in NS281 resulted in strain NS377.

Example 3: TGL3 Knockouts that Overexpress DGA1 Accumulate More Lipid than TGL3 Knockouts Alone

NS421, which contains the TGL3 knockout, and NS377, which contains the TGL3 deletion and also overexpresses R. toruloides DGA1, were grown in a 24-well plate in limiting nitrogen conditions in order to promote lipid accumulation.

Specifically, each well of an autoclaved, 24-well plate was filled with 1.5 mL of filter-sterilized media containing 0.5 g/L urea, 1.5 g/l yeast extract, 0.85 g/L casamino acids, 1.7 g/L YNB (without amino acids and ammonium sulfate), 100 g/L glucose, and 5.11 g/L potassium hydrogen phthalate (25 mM). Yeast strains that had been incubated for 1-2 days on YPD-agar plates at 30° C. were used to inoculate each well. The 24-well plate was covered with a porous cover and incubated at 30° C., 70-90% humidity, and 900 rpm in an Infors Multitron ATR shaker. After 96 hours, 20 μL of 100% ethanol was added to 20 μL of cells in an analytical microplate and incubated at 4° C. for 30 minutes. 20 μL of cell/ethanol mix was then added to 80 μl of a pre-mixed solution containing 50 μL 1 M potassium iodide, 1 mM μL Bodipy 493/503, 0.5 μL 100% DMSO, 1.5 μL 60% PEG 4000, and 27 μL water in a Costar 96-well, black, clear-bottom plate and covered with a transparent seal. Bodipy fluorescence was monitored with a SpectraMax M2 spectrophotometer (Molecular Devices) kinetic assay at 30° C., and normalized by dividing fluorescence by absorbance at 600 nm. Data was averaged in triplicate growth experiments.

NS377 accumulated approximately double the amount of lipid as NS421 showing that deletion of TGL3 in combination with DGA1 overexpression increases a cell's lipid content relative to a TGL3 deletion alone (FIG. 2).

Example 4: Cells that Overexpress DGA1 and Contain a TGL3 Deletion Accumulate More TAGs than Cells that Overexpress DGA1 but have No TGL3 Modification

NS281, which overexpresses R. toruloides DGA1, and NS377, which contains the TGL3 deletion and also overexpresses R. toruloides DGA1, were grown in 1-liter bioreactors using a high cell density fed-batch glucose process. After 118 hours, the cells were analyzed by gas chromatography to measure lipid content as a percentage of dry cell weight. Triplicate chromatography measurements were averaged for the same sample. NS377 reached approximately 4% higher lipid content than NS281, showing that deletion of TGL3 in combination with DGA1 overexpression yields better results than DGA1 overexpression alone (FIG. 3).

Example 5: Cells Comprising Genetic Modifications that Encode a Diacylglycerol Acyltransferase and Decrease the Activity of a Triacylglycerol Lipase Accumulate More TAGs

In order to test the idea that combining DGA1 and DGA2 overexpression with TGL3 deletion leads to higher lipid accumulation in Y. lipolytica, DGA2 from Claviceps purpurea was overexpressed in strain NS377. Strain NS377 contains a deletion of TGL3 and overexpresses DGA1 from Rhodosporidium toruloides as described in Example 4. DGA2 from Claviceps purpurea was selected based on prior experiments that demonstrate that this gene increases the lipid content of Y. lipolytica in combination with DGA1 from Rhodosporidium toruloides (U.S. Ser. No. 62/004,502, incorporated by reference).

FIG. 4 shows the map of pNC327, the expression construct used to overexpress C. purpurea DGA2 in NS377. The construct was linearized prior to transformation with a PacI/AscI restriction digest. The linear expression construct included an expression cassette for the C. purpurea DGA2 gene and for the BLE gene used as a marker for selection with Zeocin (ZEO). Transformants were analyzed by the fluorescent lipid assay, and the top lipid producer was designated NS432.

The lipid production of strains NS297, NS281, NS450, NS377, and NS432 were compared. A subset of these strains were either grown using a batch glucose process (in 48-well plates or 50-mL flasks) or using a high cell density fed-batch glucose process (in 1-L bioreactors). Lipid content was analyzed by fluorescence assay or gas chromatography, and strain NS432 was found to have a higher lipid content than its parent strain NS377 and the strains without the TGL3 knockout (FIG. 5). These results demonstrate the advantage of DGA1 and DGA2 overexpression in a TGL3 knockout.

INCORPORATION BY REFERENCE

All of the U.S. patents, U.S. patent application publications, foreign patents, foreign patent publications, and non-patent literature cited herein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. 

1.-13. (canceled)
 14. A method of increasing the lipid content of a cell, comprising transforming a parent cell with a first nucleic acid and a second nucleic acid; wherein said first nucleic acid increases the activity of a native diacylglycerol acyltransferase or comprises a diacylglycerol acyltransferase gene; and said second nucleic acid decreases the activity of a triacylglycerol lipase.
 15. The method of any one of claim 14, wherein said first nucleic acid comprises a diacylglycerol acyltransferase gene.
 16. The method of claim 15, wherein said diacylglycerol acyltransferase gene encodes a type I diacylglycerol acyltransferase polypeptide, a type II diacylglycerol acyltransferase polypeptide, or a type III diacylglycerol acyltransferase polypeptide.
 17. The method of claim 15, wherein said diacylglycerol acyltransferase gene encodes an amino acid sequence selected from the group consisting of: (a) an amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61; SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 85, or a biologically-active portion of any one of them; and (b) an amino acid sequence having at least 80% sequence identity to an amino acid sequence forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61; SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 83, or SEQ ID NO: 85, or a biologically-active portion of any one of them.
 18. The method of claim 14, wherein said second nucleic acid is capable of recombining with a nucleic acid sequence in a triacylglycerol lipase gene and/or a nucleic acid sequence in the regulatory region of a triacylglycerol lipase gene.
 19. The method of claim 14, wherein said second nucleic acid is capable of recombining with a nucleic acid sequence in a TGL3, TGL3/4, or TGL4 gene and/or a nucleic acid sequence in the regulatory region of a TGL3, TGL3/4, or TGL4 gene.
 20. The method of claim 14, wherein said cell is Yarrowia lipolytica or Arxula adeninivorans. 